Biology Center, Czech Academy of Sciences and Department of Molecular Biology, University of South Bohemia, Ceske Budejovice 37005, Czech Republic.

Abstract

Epithelial sheet spreading and fusion underlie important developmental processes. Well-characterized examples of such epithelial morphogenetic events have been provided by studies in Drosophila, and include embryonic dorsal closure, formation of the adult thorax and wound healing. All of these processes require the basic region-leucine zipper (bZIP) transcription factors Jun and Fos. Much less is known about morphogenesis of the fly abdomen, which involves replacement of larval epidermal cells (LECs) with adult histoblasts that divide, migrate and finally fuse to form the adult epidermis during metamorphosis. Here, we implicate Drosophila Activating transcription factor 3 (Atf3), the single ortholog of human ATF3 and JDP2 bZIP proteins, in abdominal morphogenesis. During the process of the epithelial cell replacement, transcription of the atf3 gene declines. When this downregulation is experimentally prevented, the affected LECs accumulate cell-adhesion proteins and their extrusion and replacement with histoblasts are blocked. The abnormally adhering LECs consequently obstruct the closure of the adult abdominal epithelium. This closure defect can be either mimicked and further enhanced by knockdown of the small GTPase Rho1 or, conversely, alleviated by stimulating ecdysone steroid hormone signaling. Both Rho and ecdysone pathways have been previously identified as effectors of the LEC replacement. To elicit the gain-of-function effect, Atf3 specifically requires its binding partner Jun. Our data thus identify Atf3 as a new functional partner of Drosophila Jun during development.

TheDrosophilaAtf3 protein interacts with Jun. (A) Alignment between bZIP domains of human ATF3 and Drosophila Atf3 shows identical (black shading) and similar (gray) amino acids. (B) Atf3 immunoprecipitated with Jun but not with Fos. S2 cells were transfected with Fos-His alone or together with V5-tagged Atf3; Jun was endogenous. Proteins bound to the anti-V5 antibody were analyzed by western blot (WB) with anti-V5, anti-Jun and anti-Fos antibodies. Cell lysates are shown as input. (C) Electrophoresis mobility shift assay shows binding of isolated bZIP domains of the Atf3 (A), Jun (J) and Fos (F) proteins, single or in combination, to the AP-1 and ATF/CRE consensus elements.

Genetic interaction amongDrosophila(d)atf3,junandfosduring compound eye development. The GMR-Gal4 driver was used to express the indicated transgenes in the eye-imaginal disc. jun RNAi did not affect eye morphology but was able to suppress the effect of overexpressed Atf3. Overexpression of Fos also precluded the gain-of-function effect of Atf3. Conversely, co-expression of Jun with Atf3 aggravated the phenotype.

Misexpression ofatf3causes incomplete fusion of abdominal epidermis. (A) ppl>atf3 pharate adults usually die inside puparia with a dorsally open abdominal cleft in the adult cuticle. The scar is outlined with a brown line of necrotic tissue, and it is filled with the old pupal cuticle. Other body parts metamorphose normally and the unaffected regions of the abdomen produce adult cuticle with sensory bristles. (B) Few ppl>atf3 flies eclose, invariantly bearing the scar, often with signs of bleeding. (C) Effect of atf3 expressed under the ubiquitous (arm) driver. (D) Expression of UAS-α-Catenin::GFP in posterior abdominal segments of a normally developing pupa at 27 hours APF shows that the ppl-Gal4 driver is active in LECs but not in the surrounding histoblasts that occupy the areas around and between LECs at this time. The punctate GFP signals probably come from LECs that had already been extruded and phagocytosed. (E) The Eip71CD driver is also expressed in LECs (large GFP-positive cells) but not in histoblasts. DAPI staining (magenta) shows cell nuclei. (F) Abdominal lesion in a rarely eclosing Eip71CD>atf3 adult. (G) Flp-out induction of atf3 in the polyploid larval cells reproduces the abdominal closure phenotype (for more severe defects see Fig. S4 in the supplementary material). The LECs that express atf3 and GFP persist in the adult abdomen and mainly localize to the dorsal cleft. Scale bars: 100 μm in D; 50 μm in E.

Ectopic Atf3 prevents removal and death of LECs. (A-C) In control pupae, LECs that still reside in the abdomen at 24 hours APF (A) become replaced by histoblasts. By 48 hours APF (B), histoblasts cover the entire abdomen and differentiate sensory bristles (arrowhead). The bristles have been formed and adult cuticle deposited at 72 hours APF (C). (D-I) Abdominal epidermis in ppl>atf3 pupae. Although histoblasts spread and differentiate (bristle formation indicated with arrowheads), many LECs persist, posing a barrier to histoblasts progressing from the lateral sides. The affected LECs display warping of membranes (G, arrow) and accumulate the cell junction components Dlg and DE-cadherin (arrows in F and H, respectively). (I) A large lesion affecting most of the dorsolateral abdomen of a pharate adult fly (96 hours APF) is populated by persisting LECs, the membranes of which are extremely deformed. The arrow points to a thick wall separating the scar from the adult cuticle on the left. DAPI (magenta) is used for staining nuclei; cell membranes are visualized either with anti-Dlg antibody staining (green) or by ubiquitous expression of DE-cadherin::GFP fusion protein in live pupae (G-I). Anterior is to the top in all panels. Images are z-stacks of confocal slices. Scale bars: 20 μm in A-C,F; 50 μm in D,E,G,H; 100 μm in I.

Behavior of LECs uponatf3misexpression. Images of LECs with membranes marked by DE-cadherin::GFP (ubi-shg::gfp) were captured in live pupae at indicated times. Cells expressing mRFP (magenta, bottom row) were induced to express atf3 with the flp-out system. Compared with their uninduced neighbors, which have smooth membranes (blue arrowheads), the atf3-positive LECs show membrane interdigitation (yellow arrowheads) and enrichment of DE-cadherin (arrows) on apical junctions. By 48 hours APF only Atf3-positive LECs survive, being completely surrounded and apparently pressured by histoblasts. Note that junctions between atf3-expressing neighbors are thicker compared with their boundaries with uninduced LECs or with histoblasts. Images are z-stacks of confocal slices. Scale bar: 20 μm.

LECs expressingatf3are unable to complete extrusion. Shown are images from live pupae with cell membranes marked with DE-cadherin::GFP at 24 hours APF. The flp-out system was employed to induce expression of mRFP alone (control) or together with atf3. Lines indicate three confocal cross sections, depicted below each panel. Arrowheads in control (left) point to extruding LECs that have apically constricted and detached from the epidermal layer. By contrast, although LECs expressing atf3 can apically constrict, they continue to adhere to the epithelium (arrowheads on the right) even when completely surrounded by histoblasts. Note that DE-cadherin colocalizes with the mRFP signal that marks control LECs, whereas it remains associated with the apical surface of atf3-expressing LECs that attempt to delaminate. Arrows show atf3-expressing LECs with membranes that interdigitate and contain more DE-cadherin compared with their uninduced neighbors. Scale bar: 20 μm.

Misexpression ofatf3enhances the effect ofRho1silencing and leads to mislocalization of the Rho1 protein. (A-C) Rho1 RNAi alone (A,B) caused abdominal defects similar in extent to those in ppl>atf3 animals (see ), whereas combined with misexpression of atf3 (C) it led to broader, lethal scars (compare arrows). (D) The effect of ectopic atf3 was averted by co-expression of active Rho1V14. All transgenes were induced under the ppl-Gal4 driver. (E,E′) Rho1 is redistributed in GFP-marked LECs (28 hours APF) that express atf3 upon flp-out induction. DAPI (magenta) is used for staining nuclei. Scale bar: 50 μm.

Jun is specifically required for Atf3 to disrupt abdominal development. (A-C) The severe abdominal cleft caused by misexpression of atf3 (A) was completely suppressed by Jun RNAi knockdown (B), whereas depletion of Fos had no such effect (C). Males on the left, females on the right.